A flagging system for multichannel hematology analyzers

A flagging system to identify blood specimens requiring a blood film review and/or differential leukocyte count is described. Its utility in avoiding unnecessary differential counts particularly in outpatients is given. Significant reductions in workload can be anticipated without deterioration of patient care.     

Ixodes ricinus is the dominant questing tick in forest habitats in Romania: the results from a countrywide dragging campaign

In 2010 and 2011, questing ticks were collected from 188 forested locations in all the 41 counties of Romania using the dragging method. The total of 13,771 ticks collected belonged to eleven species: Ixodes ricinus (86.9?%), Dermacentor marginatus (9.5?%), Haemaphysalis punctata (2.6?%), H. concinna (0.6?%), H. sulcata (0.3?%), H. parva (0.1?%), Hyalomma marginatum (0.02?%), D. reticulatus (0.02?%), I. crenulatus (0.007?%), I. hexagonus (0.007?%) and I. laguri (0.007?%). Ixodes ricinus was present in 97.7?% (n?=?180) of locations, occurring exclusively in 41.7?% of the locations, whereas it was the dominant species in 38.8 % of the other locations, accounting for over 70?% of the total tick community. The following most common questing ticks were D. marginatus, H. punctata and H. concinna. Ixodes ricinus co-occurred with one, two or three sympatric species. The occurrence of D. reticulatus in forested habitats from Romania was found to be accidental.     

Efficiency and cost of strategic use of acaricide for tick control in N'Dama cattle in The Gambia

The efficiency of strategic and strategic/selective applications of flumethrin spray formulation for controlling ticks were assessed, respectively, in two groups of fourteen N'Dama cattle (Group S and Group S/S) by comparison of the number of feeding ticks with thirteen untreated N'Dama cattle (Group U) over a period of 11 months (June 1996 to April 1997). During the expected peak of tick abundance, acaricide was applied fortnightly on the whole body in animals in Group S and only on the most infested body areas in cattle in Group S/S. Weight changes and skin lesions, directly associated with tick attachment, were recorded in cattle in the three groups. The costs of the two tick control schemes were estimated. Maximum level of ticks, all species together, feeding on cattle was observed in the rainy season. Both in Group S/S and Group S, cattle carried a lower (P < 0.001) number of feeding ticks than animals in Group U over the whole study period. Percentage of tick control, over the entire period of tick investigation, was satisfactory in both acaricide-treated groups, reaching 61.2 and 75.2% in Groups S/S and S, respectively. However, the proportion of control varied according to tick species or genus. Significantly lower prevalence of skin lesions was observed on the ano-genital and udder region in cattle in Group S/S (P < 0.05) and Group S (P < 0.01) in comparison with cattle in Group U. Mean amount of acaricide solution used and relative estimated cost of treatment in cattle in Group S/S were, respectively, 25- and 14-fold lower than those in cattle in Group S. At the end of the study, animals in Groups S/S and S were, respectively, 7.2 and 15.9 kg heavier than animals in Group U. The difference was statistically significant (P < 0.02) only between Groups S and U. However, the efficiency, low cost and derived benefits of the strategic/selective acaricide application scheme indicated that it might be the most cost effective.     

一种收集蜱类唾液的方法

建立了一种收集蜱类唾液的方法。注射多巴胺(20mgmL)10μL于森林革蜱DermacentorsilvarumOlenev吸血雌蜱血腔内,可使唾液腺分泌唾液,用毛细管连续收集30min,可得唾液15～20μL蜱。     

The major tick salivary gland proteins and toxins from the soft tick,Ornithodoros savignyi,are part of the tick Lipocalin family: implications for the origins of tick toxicoses

The origins of tick toxicoses remain a subject of controversy because no molecular data are yet available to study the evolution of tick-derived toxins. In this study we describe the molecular structure of toxins from the soft tick, Ornithodoros savignyi. The tick salivary gland proteins (TSGPs) are four highly abundant proteins proposed to play a role in salivary gland granule biogenesis of the soft tick O. savignyi, of which the toxins TSGP2 and TSGP4 are a part. They were assigned to the lipocalin family based on sequence similarity to known tick lipocalins. Several other tick lipocalins were also identified using Smith-Waterman database searches, bringing the tick lipocalin family up to 20. Phylogenetic analysis showed that most tick lipocalins group within genus-specific clades, suggesting that gene duplication and divergence of tick lipocalin function occurred after tick speciation, most probably during the evolution of a hematophagous lifestyle. TSGP2 and TSGP3 show high sequence identity and group terminal to moubatin, an inhibitor of collagen-induced platelet aggregation from the tick, O. moubata. However, no platelet aggregation inhibitory activity is associated with the TSGPs using ADP or collagen as agonists, suggesting that TSGP2 and TSGP3 duplicated after divergence of O. savignyi and O. moubata. This timing is supported by the absence of TSGP2-4 in the salivary gland extracts of O. moubata. The absence of TSGP2 and TSGP4 in salivary gland extracts from O. moubata correlates with the nontoxicity of this tick species. The implications of this study are that the various forms of tick toxicoses do not have a common origin, but must have evolved independently in those tick species that cause pathogenesis.     

Promotion of tick cell growth by proline and fractions from tick eggs

Tick cell lines representing three genera (Rhipicephalus appendiculatus, Dermacentor variabilis and Boophilus microplus) were grown in basal medium containing different supplements. Their effect on cell growth was measured by determining the rate at which cultures were transferred. The optimal concentration of the foetal bovine serum was 5% for the Boophilus line and 10% for the other two. Addition.of tick egg extract (TEE) increased the splitting rate 1.5–2.4 times during a period of 30 days, and changed the morphology of some cells. The crude TEE was delipidized and the protein and lipid fractions were analyzed for biological activity. The delipidized protein fraction of the TEE improved the splitting rate to about the same degree as the crude TEE. Adding l-proline to the basal medium increased the splitting rate 1.2–1.9-fold, or nearly to the same degree as did the crude TEE. The addition of the lipid and aqueous polar fractions of TEE did not improve cell growth.     

Formulation of medium for tick cell culture

We examined the effectiveness of bovine cholesterol concentrate in reducing the high level (10–20%) of fetal bovine serum (FBS) necessary to promote tick cell growth in vitro. Tick cell lines isolated from embryos ofAnocentor nitens (ANE 58),Boophilus microplus (BME 26), andRhipicephalus appendiculatus (RAE 25) were used. They were incubated in L-15 (BME 26) or L-15B (ANE 58 and RAE 25) supplemented with 10% tryptose phosphate broth (TPB), 5% (ANE 58 and BME 26) or 3% FBS, 10–90 m/ml cholesterol. A concentration of 10 g/ml cholesterol stimulated the growth rate of all three lines but more than 30 g/ml depressed growth in ANE 58 and RAE 25 cells, while multiplication of BME 26 cells was enhanced by all cholesterol concentrations tested. All three lines could be continuously grown in 5% FBS, provided that 10 g/ml cholesterol was included.Nutrients added to L-15 in the formulation of L-15B were tested singly or in combination for their ability to support tick cell growth in medium supplemented only with 5% FBS and 10 m/ml cholesterol. In L-15 alone, RAE 25 cells did not multiply. Adding glucose (Glc), glutamic acid (Glu), or -ketoglutaric acid (K) had little or no effect, and the same was true for combinations of Glc plus K, aspartic acid (Asp) plus proline (Pro) and glutamine (Gln), and minerals plus vitamins (MV). When Asp, Gln, Pro, and K were combined with Glc and/or MV and added to L-15, there was appreciable growth stimulation, but best results were obtained when Glu was also included. In this medium, i.e., L-15B with 5% FBS and 10 /ml cholesterol, lines BME 26 and RAE 25 could be continuously subcultured.     

Transgene expression and silencing in a tick cell line: A model system for functional tick genomics

The genome project of the blacklegged tick, Ixodes scapularis, provides sequence data for testing gene function and regulation in this important pathogen vector. We tested Sleeping Beauty (SB), a Tc1/mariner group transposable element, and cationic lipid-based transfection reagents for delivery and genomic integration of transgenes into I. scapularis cell line ISE6. Plasmid DNA and dsRNA were effectively transfected into ISE6 cells and they were successfully transformed to express a red fluorescent protein (DsRed2) and a selectable marker, neomycin phosphotransferase (NEO). Frequency of transformation was estimated as 1 transformant per 5000-10,000 cells and cultures were incubated for 2-3months in medium containing the neomycin analog G418 in order to isolate transformants. Genomic integration of the DsRed2 transgene was confirmed by inverse PCR and sequencing that demonstrated a TA nucleotide pair inserted between SB inverted/direct repeat sequences and tick genomic sequences, indicating that insertion of the DsRed2 gene into the tick cell genome occurred through the activity of SB transposase. RNAi using dsRNA transcribed from the DsRed2 gene silenced expression of red fluorescent protein in transformed ISE6 cells. SB transposition in cell line ISE6 provides an effective means to explore the functional genomics of I. scapularis.     

Vaccination with 'concealed' antigens for tick control

Ticks are responsible for substantial economic losses to the livestock industry, necessitating intensive use of chemical acoricides in many parts of the world. Problems of chemical residues, cost of acoricides, and development of resistance by ticks, have long been recognized and have helped to stimulate interest in tick control by immunological means (see Box 1). One approach has been to seek ways to enhance the natural immunity often acquired by animals in response to tick infestation. An alternative, discussed here by Peter Willadsen and David Kemp, is to vaccinate with 'concealed' tick antigens not normally encountered by the host, and so stimulate a different immune effector mechanism.     

Risk of predation for two reptile tick species

Experiments were conducted in the Mount Mary region of South Australia where the distributions of the two reptile-tick speciesAponomma hydrosauri andAmblyomma limbatum narrowly overlap. Engorged larvae and nymphs were placed in boxes with and without access to predators and left for 1 week. At two out of five sites there was greater loss from open access boxes in replicates during warmer periods of the year. Predation by ants appears to be the most likely explanation for the loss of ticks.     

The efficiency of patch sampling for determination of relative tick burdens in comparison with total tick counts

Quanttative data on host tick burdens ar fundamental for the initiation of control strategies and effective management of wildlife populations, but the methods of live sampling employed for domestic animals are unsuitable for sampling wild animals. Despite advances in the use of destructive methods (the scrub and digestion techniques) to obtain measures of the total tick burden on wildlife, these methods are too involved for many field workers, who often need only measures of relative tick burden. Recently, patch sampling methods have been introduced whereby only certain predilection sites are sampled, the presumption being that the number of ticks collected gives an indiccation of the relative degree of infestation. We examined the validity of patch sampling as a measure of relative tick burden by comparing adult ticks collected from the ears, head, neck, foreleg and perianal region of impala (aepyceros melampus) with total tick burdens of the same animals derived from the digestion technique. Adult ticks from patch sampling were positively and significantly correlated with total adults and total ticks (larvae, nymphs, and adults) on impala, with ticks patch sampled from the neck showing the highest correlation with the total tick burden. Comparison of relative tick loads from patch sampling with absolute tick loads from digestion for three classes of impala (females, bachelor males and territorial males) gave qualitatively similar results. We conclude that, when measures of relative tick load are sufficient and destructive sampling is not feasible, patch sampling can provide reliable information on relative tick burdens that are positively correlated with the total tick burden.     

Flagging versus dragging as sampling methods for nymphal Ixodes scapularis (Acari: Ixodidae)

The nymphal stage of the blacklegged tick, Ixodes scapularis (Acari: Ixodidae), is responsible for most transmission of Borrelia burgdorferi, the etiologic agent of Lyme disease, to humans in North America. From 2010 to fall of 2012, we compared two commonly used techniques, flagging and dragging, as sampling methods for nymphal I. scapularis at three sites, each with multiple sampling arrays (grids), in the eastern and central United States. Flagging and dragging collected comparable numbers of nymphs, with no consistent differences between methods. Dragging collected more nymphs than flagging in some samples, but these differences were not consistent among sites or sampling years. The ratio of nymphs collected by flagging vs dragging was not significantly related to shrub density, so habitat type did not have a strong effect on the relative efficacy of these methods. Therefore, although dragging collected more ticks in a few cases, the numbers collected by each method were so variable that neither technique had a clear advantage for sampling nymphal I. scapularis.